Tips for Keeping Primary Neuron Cultures Healthy

We’ve all had primary neuron cultures that have gone wrong. With so many steps in the process of obtaining and culturing these neurons, it can be difficult to pinpoint the issue. Here is a guide to help you troubleshoot your primary neuron culture at every step. 

Characteristics of a healthy culture

What should a healthy primary cortical or hippocampal neuron culture look like? In a healthy culture, neurons should adhere to the well surface within one hour after seeding, and within the first two days in culture, should have extended minor processes and show signs of axon outgrowth1,2. By four days, neurons should show dendritic outgrowth, and by one week should start forming a mature network1. Healthy primary neuron cultures should be reproducibly maintainable beyond 3 weeks3. 

What about glia?

In the brain, neurons are highly dependent on glial cells for trophic support4. This complicates neuronal culture systems since glial cells proliferate and can overgrow the neurons5. Some methods culture neurons alongside a glial feeder layer to enable trophic support without neuron overgrowth, however this can complicate the culture setup4. With Neurobasal medium and appropriate supplements, long-term neuronal cultures can be maintained long-term without feeder cells and relatively minimal glial growth3,6. If maintaining a highly pure neuronal culture with very little glial cell contamination is necessary for your experiments, use of cytosine arabinoside (AraC) is an established method to inhibit glial proliferation5. However, AraC has been reported to have off target neurotoxic effects and should only be used when necessary and at low concentrations. 

Dissection methods

Primary neuron cultures have many advantages over immortal cell lines because they are more genetically stable and more similar to post-mitotic neuronal cells3. However, this comes with the disadvantage that primary neurons must be obtained from a dissection for each new culture. If your neurons are failing to adhere or lack signs of outgrowth in the early stages following dissection, this could be an indicator of cell damage during the dissection.  

One thing to consider when optimizing your dissection process is the age of the animal. Primary neuron cultures can be generated from late embryonic to early postnatal stages, however embryonic cultures are generally preferred due to a lower density of glial cells and less defined arborization that prevents shearing of cells during dissection4,7. Because of this, E17-19 is most commonly used in rat primary neuron culture protocols3,4. If using embryonic neurons works with your experiments, consider using this timepoint to obtain healthier cells. 

Once the tissue is dissected, a key step is dissociation of the tissue. While this is commonly done using trypsin combined with mechanical trituration, it has been shown that trypsin can cause RNA degradation3,8. If you are noticing issues with the early health of your neurons, consider using papain as an alternative digestion enzyme, or for cortical neurons try dissociating by mechanical trituration alone3. Mechanical trituration to dissociate tissue should be gentle and avoid bubbles to avoid shearing by surface tension3,9. Allowing neurons to rest after dissociation can also improve the seeding process3. 

Density

Cultured neurons are happiest and healthiest when grown at a higher density. Establishing an appropriate cell density is essential for creating a culture that can differentiate and form a mature network. Ideal density depends on cell type and planned experiment; however, the following is a general guideline for rat primary neuron plating densities3:

For cortical neurons:

• Biochemistry experiments: 120 000/cm2
• Histology: 25 000 - 60 000/cm2

For hippocampal neurons:

• Biochemistry experiments: 60 000/cm2
• Histology: 25 000 - 60 000/cm2

Coating substrates

Primary neurons cannot grow directly on plastic or glass and require a growth substrate to adhere to the well surface. If your neurons are piling together into clumps and growing on top of each other, this is a sign that your coating substrate is being degraded10. The most commonly used coating substrates are poly-D-lysine (PDL) and poly-L-lysine (PLL) which are homopolymeric chains of the positively charged amino acid lysine3,4,9,11. PDL is formed from the D-lysine enantiomer and is more resistant to enzymatic degradation by proteases such as trypsin12,13. If you encounter substrate degradation issues with PLL, try replacing it with PDL. If issues with substrate degradation persist with PDL, consider switching to dPGA, an alternative growth substrate that mimics the structure of poly-lysine, but lacks peptide bonds making it highly resistant to degradation2. 

Medium and supplements

A final thing to consider when troubleshooting your primary neuron culture is medium and serum. To maintain controlled concentrations of hormones, growth factors, and nutrients, primary neuron cultures should be maintained in a serum-free culture medium6. Normal DMEM allows for glial growth, thus Neurobasal, a version of DMEM optimized for neurons, is most commonly used with added B27 supplement and L-glutamine or Glutamax3,4,6. Antibiotic supplements such as penicillin/streptomycin are typically added to control contamination, however they can alter neuronal properties such as electrical activity should only be used if they will not impact experimental outcomes14. The medium should be prepared fresh once a week with newly diluted supplements from frozen stocks9. To counteract evaporation and provide continuous nutrients, half medium changes should be conducted every 3-7 days3,4.

References

1. Dotti, C., Sullivan, C. & Banker, G. The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8, 1454–1468 (1988).
2. Clément, J.-P. et al. Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture. ASN Neuro 14, 17590914211073276 (2022).
3. Sahu, M. P., Nikkilä, O., Lågas, S., Kolehmainen, S. & Castrén, E. Culturing primary neurons from rat hippocampus and cortex. Neuronal Signal. 3, NS20180207 (2019).
4. Roppongi, R. T., Champagne-Jorgensen, K. P. & Siddiqui, T. J. Low-Density Primary Hippocampal Neuron Culture. J. Vis. Exp. JoVE 55000 (2017) doi:10.3791/55000.
5. Lesslich, H. M., Klapal, L., Wilke, J., Haak, A. & Dietzel, I. D. Adjusting the neuron to astrocyte ratio with cytostatics in hippocampal cell cultures from postnatal rats: A comparison of cytarabino furanoside (AraC) and 5-fluoro-2’-deoxyuridine (FUdR). PLOS ONE 17, e0265084 (2022).
6. Brewer, G. J., Torricelli, J. R., Evege, E. K. & Price, P. J. Optimized survival of hippocampal neurons in B27-supplemented neurobasalTM, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576 (1993).
7. Sciarretta, C. & Minichiello, L. The Preparation of Primary Cortical Neuron Cultures and a Practical Application Using Immunofluorescent Cytochemistry. in Mouse Cell Culture: Methods and Protocols (eds. Ward, A. & Tosh, D.) 221–231 (Humana Press, 2010). doi:10.1007/978-1-59745-019-5_16.
8. Vrtačnik, P., Kos, Š., Bustin, S. A., Marc, J. & Ostanek, B. Influence of trypsinization and alternative procedures for cell preparation before RNA extraction on RNA integrity. Anal. Biochem. 463, 38–44 (2014).
9. Brewer, G. J. & Torricelli, J. R. Isolation and culture of adult neurons and neurospheres. Nat. Protoc. 2, 1490–1498 (2007).
10. Thiry, L., Clément, J.-P., Haag, R., Kennedy, T. E. & Stifani, S. Optimization of Long-Term Human iPSC-Derived Spinal Motor Neuron Culture Using a Dendritic Polyglycerol Amine-Based Substrate. ASN Neuro 14, 17590914211073381 (2022).
11. McKeehan, W. & Ham, R. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J. Cell Biol. 71, 727–734 (1976).
12. Tsuyuki, E., Tsuyuki, H. & Stahmann, M. A. THE SYNTHESIS AND ENZYMATIC HYDROLYSIS OF POLY-d-LYSINE. J. Biol. Chem. 222, 479–485 (1956).
13. Li, J. & Yeung, E. Real-Time Single-Molecule Kinetics of Trypsin Proteolysis. https://pubs.acs.org/doi/epdf/10.1021/ac801365c (2008) doi:10.1021/ac801365c.
14. Bahrami, F. & Janahmadi, M. Antibiotic Supplements Affect Electrophysiological Properties and Excitability of Rat Hippocampal Pyramidal Neurons in Primary Culture. Iran. Biomed. J. 17, 101–106 (2013).